Probing the glutamate at position 304 of the allosteric site and further investigation of the proline at position 288 of the loop region of the agrobacterium tumefaciens ADP-Glucose pyrophosphorylase

ADP-Glucose pyrophosphorylase (ADPG PPase) (EC 2.7.7.27) catalyzes the rate limiting step in the glucan biosynthesis pathway. Glucans are renewable and vital carbon and energy sources making ADPG PPase an attractive target for protein engineering. Starch is a biodegradable and versatile product used in various industries. By identifying the roles of important amino acid positions, a more efficient ADPG PPase may be engineered to yield more starch.;Agrobacterium tumefaciens (Ag. t.) ADPG PPase is the only bacterial form of the enzyme with a known three-dimensional structure making it an ideal model for structure-function studies. The Ag. t. ADPG PPase enzyme is activated by fructose-6-phosphate and pyruvate and inhibited by inorganic phosphate and sulfate. These effector molecules are believed to bind at a common allosteric site. Despite the availablilty of a crystal structure (PDB 3BRK), the role of some amino acids in or near the allosteric site remains unclear. To probe glutamate at position 304, the E304A and E304D altered enzymes were generated, purified and characterized. Substitution of the E304 residue resulted in changes in allosteric behavior with the largest effects being desensitization to effectors and a lower apparent affinity for inhibitors. Previous studies indicated that the alteration from proline to aspartate at position 288 resulted in a hyperactive form of the enzyme that was completely desensitized to effector molecules. Molecular modeling suggested that the Lysine310 residue may be forming a salt bridge with aspartate at the 288site. To test this hypothesis, the K310A and P288D/K31 OA altered enzymes were generated, purified and characterized. The K310A and P288D/K310A variants displayed a large decrease in enzymatic activity, suggesting that Lysine310 may be involved in enzyme catalysis. However, while the K310A enzyme was still activated by fructose-6-phosphate and pyruvate, the P288D/K310A double mutant was desensitized to activators in a similar fashion as the P288D enzyme. These results suggest that specific salt bridge formation may not be directly involved in the hyperactivity of P288D. Further analyses of additional altered enzymes and three-dimensional structures would provide more details on the activated state of the enzyme and allow for a more accurate picture of structure-function relationships to emerge.